The present invention relates to a transmitted-illumination apparatus applicable to various types of microscopes.
There are conventional methods, such as a phase-contrast observation method, a differential-interference observation method, a modulation-contrast method and an oblique illumination method, for visualizing various colorless transparent phase-samples and observing them.
In the phase-contract observation method, a ring slit is provided at a position of a pupil of an illumination optical system of a microscope. A phase film having a conjugate shape with the ring slit is disposed at a pupil of a focusing optical system provided at a position conjugate with the ring slit.
An advantage of this observation method resides in that observation images with clear contrast can be obtained with high detection sensitivity, even for samples with a small difference in refractive index between structures, or minute granular structures of cells. On the other hand, a disadvantage of this observation method resides in that a phenomenon called xe2x80x9chaloxe2x80x9d, in which an end portion of a structure of a sample looks shining in white, occurs and this makes it difficult to determine the contour of a structure. In addition, it is necessary that the ring slit provided in the illumination optical system and the phase film disposed at the pupil plane of the observation optical system be made to coincide by projection, thereby improving the aberration performance of the pupil from the ring slit to the phase film plane. In the phase-contrast observation method, there arises no problem with the observation at a high magnification, but the aberration performance of the pupil for the observation at a low magnification or a very low magnification cannot satisfactorily be corrected. In fact, the phase-contrast observation method is applicable to objective lenses with a magnification of xc3x974 at most.
In the differential-interference observation method, two polarized light components crossing at right angles, which are produced by a birefringent crystal, are radiated on a sample plane with a slight displacement, and these light components are made to interfere with each other, thereby observing a minute structure of the sample. An advantage of this observation method resides in that stereoscopic observation with very high contrast can be performed. On the other hand, a disadvantage of this observation method is that the use of the birefringent crystal increases costs and because of use of polarized light, no exact observation image can be obtained in a case of a material which affects the polarized state. For example, a plastic Petri dish is unsuitable for the differential-interference observation. The reason is that polarized light is disturbed by birefringence of plastic material. In addition, the polarization state is disturbed by a distortion of a lens or an objective lens in the illumination optical system, a purpose-specific objective lens, etc. is needed. Moreover, since two light beams are subjected to interference, a lens capable of actual observation needs to have a magnification of xc3x974 or more, and this is not suitable for observation with a low magnification or a vary low magnification.
In the modulation-contrast observation method, as disclosed in Jpn. Pat. Appln. KOKAI Publication No. 51-128548, a slit is provided at a position of a pupil of an illumination optical system of a microscope, and a plurality of regions with different transmittances are provided at a position of a pupil of a focusing optical system. Normally, an absorption film having a proper transmittance is disposed at a region conjugate with the slit. A transmission region is provided on one side adjacent to the absorption film, and a light-shield region is provided on the other side. On a pupil plane, light transmission regions vary depending on a magnitude of refraction due to a structure in a sample, and a transmittance varies accordingly. Thus, a stereoscopic image with white/black shading can be obtained. An advantage of this observation method resides in that a stereoscopic image with shading on a phase object can be obtained with a relatively inexpensive structure. Since this method is free from halo, which occurs in the above-mentioned phase-contrast observation method, the contour of a structure can be easily observed and this method is suitable for manipulation of a cell, etc. On the other hand, a disadvantage of this observation method resides in that the detection sensitivity is lower than in the phase-contrast observation method and it is difficult to determine a minute structure. Moreover, a difficult operation for regulating the directions of the slit and absorption film needs to be performed each time the objective lens is exchanged. Besides, in order to project the slit onto the absorption film of the observation optical system, it is necessary to improve the aberration of the optical system for projecting the pupil, like the phase-contrast observation method. Because of this, with the objective lens of a low magnification or a very low magnification, the pupil aberration cannot satisfactorily be corrected and proper observation cannot be performed.
There are an oblique illumination method and a dark-field illumination method as illumination methods for visualizing phase-samples.
FIGS. 1A to 1D are schematic views of condenser lenses in general oblique illumination methods. In these figures, numeral 1 denotes an aperture stop; 2a, 2b lens groups; and 3 a sample. The aperture stop 1 limits the aperture for illumination and has a variable circular aperture. The aperture stop 1 moves in a plane perpendicular to an illumination optical axis O, thereby controlling the angle of illumination onto the sample 3. Specifically, FIG. 1B shows the state of the pupil in a case where the aperture stop 1 in the state shown in FIG. 1A has been moved and reduced. FIG. 1C shows the state of the pupil in a case where the aperture stop 1 has been further reduced. FIG. 1D shows the state of the pupil in a case where the aperture stop 1 has been shifted while being opened.
FIG. 2A is a schematic view of a condenser lens in a general dark-field illumination method. In the conventional dark-field illumination method, as shown in the figure, a stop 1a, which has an inside portion shut off and has an outside annular portion provided with a slit, is disposed near a location where an aperture stop is disposed. As is shown in FIG. 2B, the stop 1a has a central light-shield region 1b. The region 1b prevents illumination light from directly entering an objective lens. In addition, scattered light from the sample 3 is observed to realize dark-field observation. In this case, the shape of the stop 1a is selected in accordance with the numerical aperture of the objective lens, whereby dark-field observation can be made using various objective lenses.
As regards observation using microscopes, not only micro-regions but also macro-regions need to be observed. There are cases where the use of an objective lens with a magnification of xc3x971, an objective lens with a very low magnification of xc3x970.5, etc. is desired. In general, a stereomicroscope is used for observing such macro-regions. The stereomicroscope is advantageous in that the cost is low, the operability is high and stereoscopic observation can be performed. In addition, as regards illumination methods, there are means, such as dark-field illumination, bright-field illumination and oblique illumination, for visualizing transparent samples such as phase samples.
Jpn. Pat. Appln. KOKAI Publication No. 4-318804 discloses a transmission-illumination apparatus for a stereomicroscope, which permits oblique illumination. FIG. 3A shows the transmission-illumination apparatus disclosed in this publication. As is shown in FIG. 3A, this apparatus is constructed such that light from a light source 5 is guided to a mirror 8 via a collector lens 6 and a frosted glass 7, and a light beam reflected by the mirror 8 is radiated via a condenser lens 9 onto a sample 10a placed on a sample-mounting transparent member 10 and then guided to an objective lens 12. By rotating the mirror 8 and changing the angle thereof, the ratio between a dark portion 13a and a bright portion 13b of a pupil 13 of each of right and left objective lenses, as shown in FIG. 3B, can be controlled.
Jpn. U.M. Appln. KOKOKU Publication No. 41-5808 discloses a transmission-illumination apparatus for a stereomicroscope capable of selectively effecting oblique illumination and dark-field illumination. FIGS. 4A and 4B are views for describing this apparatus. As is shown in FIG. 4A, this apparatus is constructed such that light from a light source 5 is guided to a mirror 8 via a collector lens 6 and a frosted glass 7, and light reflected by a mirror 8 is radiated via a condenser lens 9 onto a sample 10a and then guided to an objective lens 12. A knife edge 15 for cutting a light beam is provided near the frosted glass 7 disposed at a position conjugate with the pupil of the objective lens 12.
As is shown in FIG. 4B, the knife edge 15 is vertically moved relative to a conjugate image 17 of the pupils of the two juxtaposed objective lenses, whereby oblique illumination and dark-field illumination is selectively effected. The aforementioned Jpn. Pat. Appln. KOKAI Publication No. 4-318804 proposes that a stop be substituted for the knife edge 15 shown in FIG. 4A.
A purpose-specific observation optical system is required for the above-described phase-contrast observation method, differential-interference observation method and modulation-contrast observation method which can perform observation of a transparent object such as a phase sample. It is also necessary, for example, to correct the optical performances of the illumination optical system and the pupil projection optical system of the observation optical system. Thus, these methods are not suitable for observation with a low magnification or a very low magnification.
In the oblique-illumination method shown in FIG. 1A, if the aperture stop 1 is shifted and reduced, as shown in FIG. 1C, the resolution and the luminance of illumination light become deficient. If the aperture stop 1 is shifted, as shown in FIG. 1D, it becomes difficult to control the degree of freedom of oblique illumination, i.e. the ratio between illumination light directly incident on the objective lens and non-incident illumination light. The reason is that the aperture stop is constructed to form a circular opening.
In addition, in the dark-field illumination illustrated in FIG. 2, the angle of dark-field illumination light varies depending on the width of the annular slit or the position of the aperture. Consequently, if the thickness, etc. of the sample varies, the sample cannot be made visible with good contrast. Specifically, in order to freely control the angle of illumination light, it is necessary to prepare many annular slits with different structures, and this is not practical.
Moreover, as regards the oblique illumination method proposed in the above-described stereomicroscope, only one of the pupils of the right and left objective lenses is illuminated. Thus, only one kind of contrast is obtained. Although the effect of oblique illumination can be obtained by disposing the slit at the pupil of the illumination optical system and thereby restricting the aperture of the pupil of the objective lens, the shape of the slit or the position of the slit is fixed in the prior art. It is thus not possible to freely and finely control the intensity of illumination light or the angle of illumination, depending on the thickness and refractive index of various samples.
As has been described above, with the conventional illumination apparatus for microscopes, phase-samples cannot satisfactorily be made visible with high contrast in observation with a low or very low magnification.
Recently, stereomicroscopes have been constructed as systems, and a wide range of magnification is required. In addition, high operability is required. In order to meet a demand for use with a wide range of magnification, it is necessary to achieve uniform illumination over a wide visual field. In view of easier use, a sample plane needs to be situated at a level as low as possible.
In the above-described prior art, the frosted glass (diffusion plate) needs to be enlarged in order to increase the visual field, and the deflecting mirror, too, needs to be enlarged. Because of this, the thickness of the illumination optical system increases, and both a demand for a wider visual field and a demand for a low-level sample plane cannot be satisfied.
Jpn. U.M. Appln. KOKOKU Publication No. 45-1105 discloses an illumination apparatus capable of performing bright-field illumination and dark-field illumination, as shown in FIG. 5. In this illumination apparatus, a light source 100 is disposed under an objective lens 101 and a sample 102. In the dark-field illumination mode, a shutter 103 is closed to shut off direct light traveling to the sample 102. In addition, light from the light source 100 is reflected by a cylindrical mirror 105 and made obliquely incident on the sample 102. In the bright-field illumination mode, the shutter 103 is opened and light from the light source 100 is made directly incident on the sample.
In this illumination apparatus, however, the light source is disposed vertical to the sample. Consequently the optical path is short and, no space is left for mounting optical members such as a filter. If an optical member is to be disposed on the optical path, the thickness of the apparatus with this structure is increased. Furthermore, since the optical path is short, the wide visual field cannot uniformly be illuminated.
An object of the present invention is to provide an illumination apparatus for a microscope, wherein a phase-sample is visualized with a good contrast without disposing a purpose-specific optical element, etc. in an observation optical system, in particular, in a region of a low magnification to a very low magnification, and a structure and a distribution thereof can be specified. Specifically, a transmission-illumination apparatus is provided wherein a contrast is successively varied for various samples with different thicknesses and refractive indices and optimal illumination is performed for the samples.
Another object of the invention is to provide a transmission-illumination apparatus wherein a sample-mounting surface can be set at a low level, that is, a height between a bottom surface of a microscope body and the sample-mounting surface can be reduced.
Still another object of the invention is to provide a transmission-illumination apparatus wherein a bright-field optical system and a dark-field optical system can be switched to observe a sample and a height between a bottom surface of a microscope body and a sample-mounting surface can be reduced.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.